Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important pathogen that causes life-threatening disease. Antibiotic treatment of EHEC infection is associated with more severe systemic disease. Thus, a deeper understanding of the virulence properties of this pathogen is needed to generate new therapeutic strategies. The potency of this pathogen stems from the combined activity of two major virulence components: Stx toxin and a complex type III secretion system (TTSS), including the associated effector proteins that are injected into mammalian cells. While each of these components was studied extensively, little is known on how these two major virulence mechanisms might interact and synergize. For example, the tissue damaging action of Stx is greatly potentiated by the host innate immune response, and Dr. Rosenshine's laboratory (as well as others) has shown that a group of type III-secreted immunomodulatory effector proteins down-regulate the inflammatory response of the host. A major obstacle in addressing the influence of immunomodulatory effectors has been the lack of a model system that recapitulates Stx activity in the context of TTSS-dependent colonization of the host. Two models recently developed by the applicants allow such studies. One employs a genetically engineered Stx-producing strain of C. rodentium, a murine pathogen that encodes a TTSS and a collection of effectors very similar to those of EHEC. This engineered C. rodentium induces a murine disease that, like human EHEC infection, features intestinal damage, renal dysfunction, progressive weight loss and death. A second model involves EHEC infection of human intestinal xenografts in SCID mice, a model that mimics human infection, particularly with respect to the documented tropism of EHEC for colonic tissue. Insights into factors that diminish or exacerbate serious Stx-mediated disease may lead to new and needed strategies to prevent the most lethal consequences of EHEC infection. As a first step towards achieving this long-term goal, we investigate an important group of effector proteins that have been shown to modulate the inflammatory response of host cells in vitro. To test whether the immunomodulatory activities of these effectors identified in vitro accurately reflect anti-inflammatory effects in vivo, and whether these effector proteins influence the delivery, dissemination and capacity of Stx to cause systemic disease, we will pursue the following specific aims: 1. Construction and in vitro characterization of CR (?stx2dact) and EHEC strains defective for production of the immunomodulatory effectors. 2. Characterization of the ability of the above set of CR (?stx2dact) strains to colonize mice and cause local and systemic Stx-mediated damage. 3. Characterization of the ability of the above set of EHEC strains to cause local and systemic Stx-mediated damage using the human intestinal xenograft model.